The invention relates to chemical, biochemical, and biological assays, and more particularly to solid phase assays for the detection of ligands.
Assays in which a sample and one or more reagents are variously reacted to form a ligand/conjugate complex such as an antibody/antigen or similar complex, which may then be observed in order to measure the presence or level of a predetermined moiety in the sample, are well known. Typically, an antibody is used to assay for the presence of an antigen for which the antibody is specific. These assays have been extended to quantitate haptens such as hormones, alkaloids, steroids, antigens, antibodies, nucleic acids, and fragments thereof, enzymes, and cell surface receptors. It is in this broad sense that the term xe2x80x9cligand/conjugatexe2x80x9d as used herein should be understood.
Sensitive immunoassays typically use tracer techniques in which a tagged constituent of the complex is incorporated, for example in the reagent, the non-complexed tagged reagent then being separated from the complexed reagent. Thereafter, the complex can be quantitated by observing a signal from the tag. Radioisotopes, fluorescent and chemiluminescent molecules, calorimetric tags, and other markers have been used to label constituents or moieties of the complex, appropriate apparatus being employed to detect and measure the radiation from the label.
In such assays where at least one component of the complex is initially bound to a solid substrate preparatory to formation of the complex, a basic problem arises because of the typically lengthy time required to bind that component to the solid substrate such as a well microtiter plate or bead, sometimes requiring incubation times on the order of hours for binding of a component to the solid phase to occur notwithstanding such expedients as heating, agitation and the like. Consequently, there is a significant amount of prior art regarding attempts to reduce this incubation time, including using microbeads, dipsticks, macrobeads, etc., but nonetheless incubation times on the order of 10 to 20 minutes are typical.
There are numerous formats for solid phase assays, but they can nonetheless be sorted into two types: sandwich and competition, both of which are well known to those skilled in the art. Sandwich assays typically require the antigen to be able to simultaneously bind to more than one conjugate. One of the conjugates is attached to the solid phase while the other conjugate is labeled with a tag. The amount of tagged conjugate attached to the solid phase is then related to the antigen concentration in a sample. A universal problem in sandwich assays is nonspecific binding, i.e., the amount of labeled conjugate that is on the solid phase, but not attached to the antigen. In designing sandwich assays, there is usually a trade-off between signal level and nonspecific binding. Increasing the concentration of labeled conjugate or incubation time of the labeled conjugate will increase the signal levels, but will also increase the amount of nonspecific binding.
Various approaches have been used to try to reduce the effect of nonspecific binding including wash buffers, detergents, blocking steps, referencing, etc., which are also well known in the art.
In competition assays a labeled moiety, either a conjugate to or an analog of the antigen, can be bound to the solid phase. The presence of antigen reduces or inhibits the binding of the labeled moiety to the solid phase. The amount of inhibition in the signal is a measure of the antigen concentration. For a competition assay to work well the amount of antigen, labeled moiety and solid phase binding sites must be roughly equal. Therefore, competition assays are usually much less sensitive than sandwich assays and also suffer from a small linear range. They are nonetheless useful for small antigens which can only bind with one conjugate at a time.
The results of chemical, biochemical, and biological assays are used to make important decisions, and therefore, the accuracy and reliability of the result is of utmost importance. Heretofore, control samples of known concentration are assayed periodically, or even simultaneously with the sample to be measured, to calibrate and verify the operation of the assay on the unknown sample. This process reduces, but does not eliminate, the possibility of error in the assay of interest.
An object of the present invention is to provide a solid phase assay method which solves many of the problems described above. Another object of the invention is to provide a xe2x80x9ccompetition-likexe2x80x9d assay with an increased linear range of determination, improved sensitivity, reduced susceptibility to errors caused by deteriorating reagents or variations in environmental conditions, reduced susceptibility to errors caused by bubbles or other mechanical problems, ease of automation, and reduced time to obtain a final result of the assay, among other things, relative to standard competition assays known in the art. A xe2x80x9ccompetition-likexe2x80x9d assay of the invention is similar to a standard competition assay except that at least one step of the competition-like assay of the invention is time limited in a manner described herein so that competitive equilibria typical of standard competition assays are not established.
Another object of the invention is to provide an improved sandwich assay in which at least one step is time limited so that problems that are associated with non-specific binding reactions and are typical of standard sandwich assays are avoided.
Yet another object of the invention is to eliminate time-consuming incubation steps in a solid phase assay method or to significantly reduce the required time to run the assay.
The above objects are accomplished by the present invention which comprises a method of performing binding assays, utilizing a solid phase, in which at least one or more of the analyte and/or ligand components is or are allowed only a limited contact time with the solid phase component. In the present methods, the solid phase material is preferably coated with a substantial excess of binding ligand. In the preferred embodiments, as described in our co-pending, commonly assigned U.S. patent application Ser. No. 07/924,720, the contact time of the solid phase component with the analyte-containing sample of the reagent-containing sample is limited by means of flowing the sample relatively rapidly past the solid phase material. The present invention comprises several variations and improvements, described in more detail below, each of which includes the limited solid phase contact time.
The present invention comprises methods of detecting the presence or level of an analyte in a sample by detecting the formation of a binding complex on a solid phase. Preferred xe2x80x9ccompetition-likexe2x80x9d methods of the invention comprise the steps of:
(a) mixing the sample with a second ligand capable of binding with said analyte so that an analyte/second ligand complex is formed;
(b) contacting the mixture produced in step (a) with a solid phase having bound thereto a first ligand capable of binding with the second ligand so that a first ligand/second ligand complex is formed, the contacting being performed under conditions and for a time sufficiently limited that dissociation of the analyte/second ligand complex formed in step (a) is substantially inhibited;
(c) binding a detectable tag to the second ligand-either prior to or after step (a) or step (b) so that a portion of the tag is retained on the solid phase upon formation of the first ligand/second ligand complex;
(d) detecting the portion of the tag to detect formation of the first ligand/second ligand complex on the solid phase, so that the presence or level of the analyte in the sample can be determined.
As will be readily appreciated by one of ordinary skill in the art, such competition-like assays could also be used to quantify the binding constant for a particular binding ligand such as, for example, an antibody.
Preferred xe2x80x9csandwich-typexe2x80x9d methods of the invention comprise the steps of:
(a) contacting the sample with:
(i) a solid phase having bound thereto a first ligand capable of binding the analyte; and
(ii) a second ligand capable of binding to the first ligand or to a first ligand/analyte complex so that a first ligand/analyte/second ligand complex is formed on the solid phase, the contacting being performed under conditions and for a time sufficiently limited that any non-specific binding between the second ligand and the solid phase is substantially inhibited;
(b) binding a detectable tag to the second ligand either prior to or after formation of the first ligand/analyte/second ligand complex so that a portion of the tag is retained on the solid phase upon formation of the first ligand/analyte/second ligand complex;
(c) detecting the tag to determine the presence or level of the analyte in the sample.
The invention also provides methods for single-point calibration and quality assurance that can be used in conjunction with the above-mentioned competition-like and/or sandwich-type assays.
The present invention involves substantially increasing the sensitivity of standard competition-type and sandwich-type immunoassays by a process wherein at least one or more of the analyte and/or ligand components is flowed over the surface of and contacts a solid phase device (e.g. beads, a capillary tube interior surface, a microtiter plate or other device), the solid surface having been suitably coated or impregnated in advance with a binding partner of the analyte and/or ligand.
As described hereinafter, in some cases the analyte-containing fluid is pre-reacted with a label or a labelled binding partner for the analyte. In other instances, after the analyte-containing sample is flowed over and contacts the solid surface, at least one additional solution containing a tagged second binding partner, a tagging agent or a tag component is also flowed over and contacts the solid surface. The solid surface may then be washed with a suitable washing solution and the signal intensity of the tag immediately measured.
Alternately, as in the preferred embodiment described in the following paragraph, the accumulation of label on the solid phase material may be monitored continuously in real time. In this case, in at least some assays, the wash step is unnecessary as the rate of accumulation of signal may be successfully used to accurately quantify analyte.
In the preferred embodiment, the assays of this invention may be conducted in a system comprising the flow cell system described in co-pending U.S. patent application Ser. No. 07/924,720. In preferred embodiments of the system, at least one separate mass of discrete beads coated with a binding partner, is disposed within a cylindrical capillary conduit, which is preferably transparent and is arranged within and passes through the focal region of a focusing lens means. The cylindrical conduit is positioned transversely to the optical axis of the lens means and behind the center of curvature of the lens means. As already noted, the assays of this invention can also be conducted in various other systems, typically with lower sensitivity as hereinafter described, including immunologically coated capillaries, microtiter plates, etc.
In this system, when the coated mass of beads or the like is translucent and the capillary is translucent or transparent, the development of fluorescence can be monitored and measured as the assay proceeds, using optical measuring equipment and an electrical detector means as proposed in U.S. patent application Ser. No. 07/924,720. Measurement of tagged ligand/conjugate complexes bound to other solid surfaces can be made, depending upon the nature of the tag, using measuring means heretofore utilized in such measurements.
The assays of this invention, in general, contemplate coating the solid surface with a substantial excess of a binding partner (for sandwich assays) or analog (for competition-like assays) of the analyte, flowing the analyte-containing fluid and/or any other fluids over and in contact with the solid surface at a rapid rate and under conditions which expose the surface area of the solid to the analyte- or other reactant-containing fluid to the maximum possible extent.
It is believed that a key element of the invention is that the contact time of the analyte and reagent containing solutions and the solid phase material be relatively short. For example, in the preferred embodiment, only a relatively small volume of the analyte (about 2 microliters) is in contact with the solid phase material at any given time.
For competition-like assays of the present invention, xe2x80x9ccontact timexe2x80x9d refers to the average amount of time that an individual analyte/second ligand complex is in contact with the solid phase. At typical flow rates, each xe2x80x9cincrementxe2x80x9d of pre-mixed sample (where xe2x80x9cincrementxe2x80x9d refers to a volume containing, on average, a single analyte/second ligand complex) is in contact with the solid phase for less than about one minute, preferably less than about ten seconds, and most preferably less than about one second. For example, at a flow rate of 1000 ul/min, each increment of pre-mixed sample is typically in contact with the solid phase for only approximately one five-hundredth of a minute.
For improved sandwich assays of the present invention, xe2x80x9ccontact timexe2x80x9d refers to the total amount of time that second ligand is in contact with the solid phase.
While the optimum contact time may vary with other assay parameters including the binding kinetics of the particular analyte being studied, it is anticipated that the optimum contact time in all cases will be relatively short.
The assays of this invention are applicable to a wide range of analytes including monovalent and polyvalent entities. These assays can be directly conducted successfully on suitably diluted samples of fluids heretofore considered difficult to assay directly, such as, e.g., whole blood, milk, etc.
The assay methodology of this invention has numerous advantages over the methodology conventionally used in the art. These advantages include, but are by no means limited to, faster running time, greater sensitivity especially at low analyte concentrations, elimination in many cases of incubation time and diminution thereof in all cases, ease of automation, and high reproducibility, and in the particular case of competition assays, extended linear range.
As described below, using the present assay methodology, one may establish a specific performance curve for each analyte whereby false negatives and false positives due to abnormally high levels of binding and erratic results caused by performance errors can be readily eliminated.
Using the methodology of the present invention, single point calibration with a fluid of known antigen concentration can be utilized for competition-like assays as well as those of the sandwich type. This is particularly useful in eliminating false negatives and false positives due to abnormally low binding levels (e.g., such as may be caused by the presence of inactive antibodies). Other and further advantages and benefits of the present invention are discussed hereinafter or will be apparent from the detailed discussion below.